Spermatogenesis is the process by which immature male germ cells, through a complex series of events involving mitosis, meiosis, and differentiation of distinct spermatogenic cell types, are transformed into mature spermatozoa capable of fertilizing an ovum. Each spermatogenic cell type displays unique patterns of gene expression which ensure the production of proteins important for the specialized functions of these cells. The long- term goals of this project are to elucidate the mechanisms which regulate gene expression in spermatogenic cells and to understand the functions of regulated gene products in these cells. As a means to realize these goals, we propose to study the expression of Heat Shock Transcription Factor 2 (HSF2) in spermatogenic cells and its function in regulating hsp gene expression in these cells. Previous studies indicate that expression of several members of the hsp70 and hsp90 gene families (hsp70.2, hsc70t, hsp86) is regulated in spermatogenic cells, and that the promoters of these genes contain heat Shock Elements (HSEs), the HSF2 recognition sequence. Our previous studies demonstrate that HSF2 expression is regulated in germ cells, that the HSF2 protein exhibits constitutive DNA-binding activity in testis, and that HSF2 interacts with sequences in the hsp70.2 gene promoter. The hypothesis to be tested is that HSF2 expression and activity is regulated in germ cells to control hsp gene expression. We propose to precisely define the spatial and temporal patterns of HSF2 mRNA expression in testis and identify the mechanism which regulates HSF2 mRNA levels in testis cell types. HSF2 function in regulating gene expression in germ cells will be explored by determining the correlation between cellular localization of active HSF2 protein and hsp gene expression , by examining HSF2 interactions with hsp gene promoter sequences, both in vivo and in vitro, and by inhibiting HSF2 expression in germ cells and measuring resulting alterations in hsp gene expression. A second, related hypothesis to be tested is that the cellular stress response, which is mediated by Heat Shock Transcription Factor 1 (HSF1) and functions to protect cells from harmful effects of elevated temperature on cellular proteins, is involved in the well-known inhibitory effects of heat o spermatogenesis . This occurs either because spermatogenic cells lack the stress response and so are sensitive to heat-induced loss of protein function, or because stress response induction disrupts normal patterns of gene expression, thus interfering with essential cellular functions. To test this hypothesis, the stress-responsiveness of spermatogenic cells will be measured using probes for the three major parameters of the stress response; HSF1 DNA-binding activity, hsp70 mRNA, and hsp70 protein. The proposed studies will increase our understanding of the mechanisms of gene regulation in spermatogenic cells and of the importance of regulated gene expression for the specialized functions of these cells, and will also contribute to our understanding of how elevated temperature inhibits spermatogenesis. This information will provide a framework for exploring disease processes which affect spermatogenesis, and for development of new male contraceptives.